The movement of lymphocytes from the blood into secondary lymphoid organs, such as lymph nodes, and back to the blood again is known as lymphocyte recirculation. This process is an essential component of immune surveillance, as it maximizes the exposure of the body's repertoire of lymphocyte specificities to antigens that become sequestered and processed within secondary lymphoid organs. A primary step in the migration of blood-borne lymphocytes into a lymphoid organ is their highly specific adhesion to the specialized endothelial lining of postcapillary venules known as high endothelial venules or HEV. Lymphocyte adherence to HEV in lymph nodes is mediated by a lymphocyte surface molecule known as gp90MEL, which was initially identified in the mouse by an adhesion blocking monoclonal antibody, MEL-14. As revealed by its cloning, gp90MEL is a type I transmembrane protein consisting of calcium-dependent lectin domain, an EGF-like domains, two complement-regulatory domains, a transmembrane domain, and a short cytoplasmic tail. gp90MEL, now commonly referred to as LECAM-1, is a member of the newly emerging "selectin" or "LECAM" family of cell-cell adhesion proteins, which also includes ELAM-1 and GMP-140/PADGEM. As a consequence of their amino-terminal lectin domains, all of these proteins function as calcium-dependent lectins. Moreover, all recognize sialylated ligands. In the case of LECAM-1, biological ligands from lymph node HEV have been identified as two sulfated, sialylated, and fucosylated O-linked glycoproteins, known as Sgp50 and Sgp90. The focus of the present grant proposal is the biochemical and functional characterization of these ligand molecules. The specific aims are as follows: 1) using a soluble recombinant form of LECAM-1 as the basis for an affinity matrix, to isolate chemical quantities of the ligands from bovine lymph nodes; 2) with polyclonal and monoclonal antibodies prepared against the ligands, to confirm that the ligands are localized to lymph node HEV and serve as adhesion sites for lymphocytes; 3) to determine whether one or both of the ligand molecules can be reconstituted as adhesion molecules on a plastic substratum; 4) in collaboration with Genentech, to clone the polypeptides of the ligand molecules; 5) at the biochemical and molecular levels, to study the biosynthesis and cytokine regulation of the ligand molecule in a cell culture system of HEV endothelial cells; 6) to determine by metabolic radiolabeling techniques, the carbohydrate structures of the ligand oligosaccharides and to define the minimal carbohydrate recognition determinants of these carbohydrates; and 7) based on the knowledge that LECAM-1 on neutrophils mediates the rolling interaction of neutrophils with postcapillary venules at inflammatory sites, to identify the venular ligand for LECAM-1 and to compare them structurally to the HEV-ligands. From these studies a detailed biochemical understanding is likely to emerge of a critical cell adhesion event which is important not only in the normal trafficking of lymphocytes in the body but also in the targeting of leukocytes to inflammatory sites.